In situ hybridization method

ABSTRACT

A method of identifying the presence of a known target sequence in nucleic acid contained in a fixed cellular or subcellular biological structure. By adding a stable, reporter-labeled RecA/single-stranded probe complex to the cellular or subcellular structure, the target sequence can be effectively labeled by in situ hybridization, allowing the target sequence to be visualized histologically and microscopically or detected by in situ cytometry or cell sorting flow techniques.

This application is the national stage of PCT/JP92/01128, filed on Sep.2, 1992, which a continuation-in-part of co-owned, U.S. application Ser.No. 07/755,291, filed 4 Sep. 1991.

1. FIELD OF THE INVENTION

The present invention relates to a diagnostic method for performing insitu hybridization with double-stranded DNA targets.

2. REFERENCES

Alexandrov, S. P. M., et al., Chromosoma, 96:443 (1988).

Baan, R. A., et al., Prog Clin Biol Res 340A:101 (1990).

Blum, H. E., et al., Lancet, 771 (1984).

Blum, H. E., et al., Virology, 139:87 (1984).

Buchbinder, A., et al., J of Virol Methods, 21:191 (1988).

Chen, T. R., Cytogenet Cell Genet 48:19 (1988).

Cheng, S., et al., J. Biol. Chem. 263:15110 (1988).

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Disteche, C. M., et al., Cytometry 11:119 (1990).

Emmerich, P., et al., Exp Cell Res 181:126 (1989).

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Griffith, et al., Biochem. 24:158 (1985).

Haase, A. T., et al., Virology, 140:201 (1985).

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Joseph, A., et al., Exp Cell Res, 183:494 (1989).

Keller, G. H., et al., Anal. Biochem, 170:441 (1988).

Kitazawa, S., et al., Histochemistry, 92:195 (1989).

Korba, B. E., et al., Virology, 165:172 (1988).

Korenberg, J. R., et al., Cell, 53:391 (1988).

Lawrence, J. B., et al., Cell, 52:51 (1988).

Lawrence, J. B., Genome Analysis, 1:1 (1990).

Lebo, R. V., et al., Science, 225:57 (1984).

Lichter, P., et al., Science, 247:64 (1990).

Lichter, P., et al., Nature, 345:93 (1990).

Lucas, J. N., et al., Int J Radiat Biol, 56(1):35 (1989).

Madiraju, M., et al., Proc. Natl. Acad. Sci. USA, 85:6592 (1988).

McCormick, M. K., et al., Proc. Natl. Acad. Sci. USA, 86:9991 (1989).

Meyne, J., et al., Genomics 4:472 (1989).

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Narayanswami, S., et al., Cytometry, 11:144 (1990).

Niedobitek, G., et al., Am J of Pathology, 131(1):1 (1989).

Noonan, C. A., et al., Proc Natl Acad Sci USA, 83:5698 (1986).

Ono, Y., et al., Nucleic Acids Res. 11:1747 (1983).

Pinkel, D. et al., Proc Nat Acad Sci, 83:2934 (1986).

Shen, D., et al., Cancer Research, 48:4334 (1988).

Shibata, T., et al., J. Bio. Chem., 256:7557 (1981).

Simon, D., et al., Cytogenet Cell Genet, 39:116 (1985).

Trask, B., et al., Hum Genet 78:251 (1988).

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Weier, H., et al., BioTechniques 10(4):498 (1991).

Zischler, H., et al., Hum Genet, 82:227 (1989).

3. BACKGROUND OF THE INVENTION

In situ hybridization employs direct hybridization of a DNA probe withDNA or RNA in biological structures, typically permeabilized cells,subcellular fractions, or fixed chromosome preparations. Because themethod can yield morphological information about the localization ofspecific-sequence target nucleic acid(s) in fixed biological structures,it is applicable to many areas of biomedical research, such asdevelopmental biology, cell biology, genetics and particularly gonemapping, pathology and gone diagnostics.

In most applications, in situ hybridization is directed toward a targetsequence in a double-stranded duplex nucleic acid, typically a DNAduplex associated with a pathogen or with a selected sequence in viralor cell chromosomal DNA. In this method, as it has been practicedheretofore, a single-stranded labeled probe is added to thepermeabilized structure, which has been heated to a temperaturesufficient to denature the target duplex nucleic acid, and the probe anddenatured nucleic acid are allowed to react under suitablehybridization, or reannealing conditions. After removal of unbound(non-hybridized) probe, the structure is processed for examination forthe presence of reporter label, allowing the site(s) of probe binding totarget duplex nucleic acid to be localized in the biological structure,i.e., in the context of cell or subcellular morphology.

The method has been widely applied to chromosomal DNA, for mapping thelocation of specific gene sequences, and distances between known genesequences (Lichter, Meyne, Shen), for studying chromosomal distributionof satellite or repeated DNA (Weier, Narayanswami, Meyne, Moyzis,Joseph, Alexandrov), for examining nuclear organization (Lawrence,Disteche, Trask), for analyzing chromosomal aberrations (Lucas), forlocalizing DNA damage in single cells or tissue (Baan) and fordetermining chromosome content by flow cytometric analysis (Trask).Several studies have reported on the localization of viral sequencesintegrated into host-cell chromosomes (e.g., Harders, Lawrence, Lichter,Korba, Simon). The method has also been used to study the position ofchromosomes, by three-dimensional reconstruction of sectioned nuclei(van Dekken), and by double in situ hybridization with mercurated andbiotinylated probes, using digital image analysis to study interphasechromosome topography (Emmerich).

Another general application of the in situ hybridization method is fordetecting the presence of virus in host cells, as a diagnostic tool(Unger, Haase, Noonan, Niedobitek, Blum). In certain cases where thenumber of virus particles in the infected cell is very low, it may benecessary to first amplify viral sequences by in situ adopted polymerasechain reaction (PCR) methods (Haase, 1990, Buchbinder).

The in situ hybridization method described above has a number oflimitations. The most serious limitation is the requirement fordenaturing the duplex target DNA, to form the necessary single-strandedform of the target. Denaturation typically is performed by heating thesample or treating with chemicals and heat. The heat treatment canproduce spurious and unwanted changes in the nucleic acid beingexamined, related to structural changes and nucleic acid reassociationwith repeated sequences within the DNA. The repeated DNA sequences canrandomly reassociate with one another. The step also adds to the timeand effort required in the method.

Secondly, where the target sequence of interest is present in very lowcopy number, the method is limited, by renaturation kinetics, to longrenaturation times. Even then, the method may be incapable of producingprobe/target renaturation events at low target concentration. Thislimitation may be partly overcome, as indicated above, by firstamplifying the target duplex in situ by modified PCR methods. However,the PCR approach involves additional steps, and may be unsuitable formany in situ studies, such as those involving localization of genomicchromosomal DNA sequences.

4. SUMMARY OF THE INVENTION

It is therefore a general object of the present invention to provide anin situ hybridization method, for use in detecting and/or localizingtarget nucleic acid, typically duplex DNA, in a fixed biologicalstructure, which (a) does not require heat denaturation of the targetduplex, and (b) is not limited in target duplex copy number byrenaturation kinetics.

The present invention includes a method of identifying the presence of aknown target sequence in a double-stranded nucleic acid contained in acellular or subcellular biological structure, in a specificmorphological relationship with the structure. The method includesadding to the structure, a probe complex composed of RecA protein stablybound to a single-stranded, reporter-labeled nucleic acid probe which iscomplementary to one of the strands of the duplex target sequence, underconditions in which the complex can contact the duplex nucleic acid. Thecomplex is allowed to bind to the target sequence under non-denaturingconditions. After removing unbound complex, the structure is examinedfor the presence of the reporter-labeled probe bound to the nucleicacid.

The complex is preferably stabilized by preparation in the presence ofATPγS. The probe may be labeled with a detectable reporter, such as aradiolabel, enzyme or fluorescence tag, or with a ligand, such as biotinor digoxigenin, which can be subsequently reacted with a reportermolecule specific for the ligand, and carrying a detectable reporter.The complex may also be stabilized using other cofactors including, butnot limited to, ATPγS, GTPγS, ATP, dATP and a combination of ATPγS andADP.

In one general application, the method is used for detection andlocalization of genomic sequence(s) in fixed chromosome DNA structure(s)in metaphase spreads. In one embodiment, the microscopic ultrastructureof the chromosomes is determined, for example, by fluorescencemicroscopy, using fluorescence banding patterns. The location of thebound complex in relation to the known ultrastructure is then determinedindependently, for example, by a fluorescence-labeled probe complexwhose fluorescence excitation wavelength is different from that of thechromosome banding fluorescence. Alternatively, fixed cells or cellularstructures are probed in suspension followed by flow cytometric ormicroscopic analysis.

In another general application, the method can be used for detecting thepresence of virus or integrated virus-specific genomic sequences in ahost cell. The binding of a fluorescence-labeled probe to the virussequence may be determined by fluorescent microscopy or fluorescenceactivated cell sorting (FACS) or a light or fluorescent or laserscanning microscope. Where an enzyme label is used a light microscopecan be used to visualize colored (e.g., black) peroxidase or alkalinephosphatase product produced by the reporter enzyme.

Another embodiment of the present invention includes a method ofidentifying the presence of a known viral nucleic acid target sequencecontained in a fixed cellular or subcellular biological structure. Suchknown viral nucleic acid targets include known DNA viruses (such ashepatitis B virus) or RNA viruses that can have a detectable duplexnucleic acid phase in their life cycle. In this method, the fixedstructures or substructures can be incubated in 10 mM Tris-acetatebuffer, pH7.5, at 55°-60° C. before the addition of the RecA probecomplex in order to increase reaction efficiency: this step does notdenature the cellular DNAs.

The present invention also includes a method of detecting a single copynucleic acid sequence, typically a duplex DNA sequence, contained in acellular or subcellular biological structure. In this method thecellular or subcellular biological structure(s) are fixed. A probecomplex (composed of RecA protein stably bound to a single-stranded,reporter-labeled probe which is complementary the single-copy nucleicacid target sequence) is added to the cellular structure or substructureunder conditions in which the complex can contact the nucleic acidtarget sequence. The complex is then allowed to bind to the targetsequence under non-denaturing conditions. Unbound complex is thenremoved from the structure and the structure is examined for thepresence of the reporter-labeled probe bound to the nucleic acid.

In this method of single-copy nucleic acid detection, the cellularstructures or substructures can be fixed and analyzed in solution or onslides. The fixing can also include incubatation of the fixed structuresor substructures in 10 mM Tris-acetate buffer, pH7.5, at 55°-60° C. Inthe method of the present invention, the complex can be bound to thetarget sequence under non-denaturing conditions in reactions carried outfor less than 2 hours.

The method of the present invention can also include the addition ofaccessory proteins, such as single-strand binding protein (SSB),topoisomerase I or topoisomerase II.

The present invention also includes kits containing components useful tocarrying out the methods described above. One example for a kit for insitu detection of a known viral nucleic acid in a sample may include (i)a probe derived from the viral DNA sequences, (ii) RecA proteineffective for coating the probe, and (iii) means of detecting thebinding of the probe to the known viral DNA in a sample. Such kits mayalso include RecA-protein coated DNA.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are fluorescence photomicrographs of chromosome X alphasatellite DNA probe used for detection of decondensed or partiallydecondensed alpha satellite chromosomal centromeric DNA target sequencesin native, nondenatured (1A) and heat-denatured (1B) methanol-aceticacid fixed interphase HEp-2 cell nuclei;

FIGS. 2A and 2B are fluorescence photomicrographs of alpha satellite DNAprobe to chromosome 7 used for detection of decondensed chromosomalcentromeric DNA target sequences in native, nondenatured (2A) andheat-denatured (2B) fixed nuclei in interphase HEp-2 cells;

FIGS. 3A and 3B are photomicrographs taken under fluorescence microscopy(3A) and phase microscopy (3B), at the same focus, showing thedistribution of chromosome X alpha satellite DNA in a dividing fixedHEp-2 cell nucleus.

FIGS. 4A-4D illustrate steps for gene localization on a chromosome,employing the method of the invention;

FIGS. 5-10 show various types of chromosomal aberrations (upper framesA), and the corresponding fluorescence pattern which would be seen withsuch aberrations (lower frames B); and

FIGS. 11A-11C illustrate the steps in detecting virus infection ofcells, by fluorescence activated cell sorting, in accordance with theinvention.

FIG. 12 presents a photograph of a cell preparation showinghybridization signal from fixed HEp-2 metaphase chromosomes hybridizedwith RecA-coated, biotinylated, nick-translated probe to humanchromosome 1 alpha-satellite centromeric sequences.

FIGS. 13A to 13F show RecA-mediated native fluorescence in situhybridization detection of unique p53 chromosome 17 tumor suppressorgene sequences in ATCC HEp-2 and HCC "Alexander" cells in suspension.

FIGS. 14A to 14D show RecA-mediated native fluorescence in situhybridization detection of unique p53 gene sequences in ATCC HEp-2 cellnuclei on slides.

FIGS. 15A to 15E show RecA-mediated native fluorescence in situhybridization detection of Hepatitis B Virus (HBV) nucleic acidsequences in ATCC HCC "Alexander" cells in suspension.

FIGS. 16A to 16C show specificity of HBV target detection usingRecA-mediated native fluorescence in situ hybridization detection inhuman HCC cells tested by competition hybridization.

6. DETAILED DESCRIPTION OF THE INVENTION

I. In situ Hybridization Method

This section describes the basic methodology of in situ hybridization,in accordance with the invention, as applied to various biologicalstructures containing a duplex DNA target with a repeated or uniquespecific basepair sequence.

A. Preparation of Biological Structures for DNA Detection

The method of the invention is designed for detecting, bycomplementary-basepair hybridization, a selected target sequence in abiological structure contain a duplex nucleic acid, usually a DNA/DNAduplex nucleic acid. The biological structure is any morphologicallydistinct structure, such as a cell, sperm, parasite, subcellularfraction or chromosomal preparation containing the target nucleic acid.

The target duplex in the structure is typically chromosomal DNA, ornucleic acid duplex material associated with a viral, parasitic orbacterial pathogen, such as virus particles composed of viral duplexgenome encapsulated or released from being encapsulated in viral coatproteins. Methods of preparing fixed biological structures, such ascells, nuclei, and chromosomal preparations generally follow those usedin conventional in situ hybridization by DNA duplex denaturation andreannealing.

Briefly, the cellular compartment and DNA structure may be further fixedor permeabilized by treatment with an organic solvent and acid orcross-linking agent to fix the structural components in their naturalmorphological relationship. Common fixatives include acetic acid, salts,methanol, formalin, paraformaldehyde, and glutaraldehyde. Afterfixation, tissue sample may be prepared for slide presentation byembedding in wax or by freezing, followed by sectioning into thinslices.

More generally, the biological material is treated with one or more of anumber of agents capable of deproteinizing and/or delipidizing thestructures. Such methods can involve the use of proteases, lipases,acid, organic solvents including alcohols, detergents or heatdenaturation or combinations of these treatments. A common treatmentinvolves one or more washes with methanol:acetic acid.

Other pretreatments may be useful in reducing background, such as use ofinhibitors of non-specific binding of nucleic acids. For example,prehybridization with non-specific carrier DNA (e.g. salmon sperm) orRNA (e.g. tRNA), may act to reduce non-specific probe binding to thefixed DNA-target structure.

Cellular structures of interest may be individual cells, obtained forexample from cell culture, cells present in a tissue section or bodyfluid. Typically, cellular structures from a tissue are sectionedcryogenically, then treated on a slide, as above, to permeabilize thesection, such as by treatment with methanol:acetic acid. Cellularstructures may be studied to determine intracellular localization ofgenomic target sequence(s), or for detecting the presence and/orlocalization of an infective organism, such as virus, bacteria, orparasite in the cells.

Subcellular structures, such as nuclei and mitochondria, can be preparedby conventional fractionation methods, such as isopycnic centrifugation,to obtain subcellular material in enriched or substantially purifiedform. Thereafter, the enriched structure preparation may bepermeabilized and deproteinized, as above, probed either in solution oraffixed to a slide, as by drying.

Alternatively, the cells may be pretreated with 75 mM KCl, followed bytreatment with methanol:acetic acid, to remove cytoplasm. This fraction,after purification may be further treated for probe hybridization. Thismethod is illustrated in Examples 3-4 and 12-14 for the preparation ofHEp-2 cell nuclei for in situ hybridization.

Briefly in these examples, HEp-2 cells were pelleted by low-speedcentrifugation and the pellet was resuspended in 75 mM KCl for between 5and 15 min for a desired amount of nuclear swelling to occur, followedby addition of ice cold methanol:acetic acid and centrifugation. Aftergeneral further addition of ice cold methanol:acetic acid and gentleagitation of the cells after each addition followed by centrifugation,cytoplasm was degraded from the nuclei. The resulting isolated nucleipreparation was resuspended in methanol:acetic acid, placed in 10 μlaliquots on microscope slides, dried, and the slides stored at -20° C.for later use. Alternatively, cells can be harvested using standardconditions, washed in 1× phosphate buffered saline (PBS) and fixed in100% methanol or 70% ethanol then stored at -20° C.: these cells can beused in solution hybridization detection reactions.

Another structure of general interest is a fixed chromosome preparation,typically derived from cells in metaphase (Pinkel, Cherif). Thepreparation may contain the entire set of genomic chromosomes from thecell, such as the preparation in FIGS. 1A and 1B, or individual,isolated chromosomes, such as can be obtained by published methods(Lebo, McCormick) or chromosome fragments. The chromosomes are generallytreated with methanol:acetic acid, placed on a slide, then affixed tothe slide with drying.

A variety of other subcellular structures, such as mitochondria, orpathogenic structures including parasites isolated from cell or bloodsamples, such as virion particles, may also be prepared according tostandard methods, and fixed and permeabilized for in situ hybridizationas above.

B. Target-Specific DNA Probe

The probe used in the method is a single-stranded nucleic acid, usuallya DNA strand probe, or derived by denaturation of a duplex probe, whichis complementary to one (or both) strand(s) of the target duplex nucleicacid. The probe sequence preferably contains at least 90-95% sequencehomology with the target sequence, to insure sequence-specifichybridization of probe and target. The single-stranded probe istypically about 100-600 bases long, although a shorter or longerpolynucleotide probe may also be employed.

The probe may be constructed or obtained by one of a number of standardmethods. Many probes, such as various satellite DNA sequences arecommercially available in single-stranded or double-stranded form. Otherprobes can be obtained either directly from viruses, plasmids andcosmids or other vectors carrying specific sequences, or, if desired, byrestriction digest of the source of the probe DNA, such as a vector,followed by electrophoretic isolation of specific restriction digestionfragments. Probes obtained in this manner are typically indouble-stranded form, but may, if required, be subcloned insingle-stranded vectors, such as an M13 phage vector.

Alternatively, the probe may be prepared in single-stranded form byoligonucleotide synthesis methods, which may require, for larger probes,forming subfragments of the probe, then piecing the subfragmentstogether.

The probe is labeled with a reporter or ligand or moiety which allowsdetection of the targeted sequence in situ. For autoradiographicdetection, the reporter is a radiolabel, such as ³² P-labeled probeformed, for example by nick translation or polymerase chain reaction inthe presence of labeled nucleotides.

For fluorescence detection, the probe may be labeled with one of aselection of fluorescence groups, such as FITC, BODIPY, Texas Red, orCascade Blue which is excitable in a specific wavelength, such as 490,540, and 361 nm. The groups are derivatized to 3' or 5' probe ends or byincorporation or reaction at internal positions, according to standardmethods (Urdea, Keller, Zischler).

Alternatively, the probes may be labeled with a ligand-type reporter:such as biotin (Weier), digoxigenin (Zischler), or bromodeoxyuridine(BrdUrd) or other modified bases including fluorescein-11-dUTP(Boehringer-Mannheim) (Kitazawa). The probe reporter groups aredetected, in situ, by reaction of the hybridized probe with a secondaryreporter molecule which (a) binds specifically and with high affinity tothe probe ligands, and (b) contains a detectable reporter. The bindingmoiety of the secondary molecule may be avidin or streptavidin, forbinding to biotinylated nucleotides, anti-digoxigenin antibody, forbinding to digoxigenin-labeled nucleotides, and anti-BrdUrd antibody forbinding to BrdUrd-labeled probe.

The detectable reporter in the secondary molecule is typically afluorescence label, but may also be a radiolabel, for autoradiographicdetection, an antibody, an enzyme, for colorimeteric orchemiluminescence detection in the presence of a suitable substrate, orcolloidal gold (Narayanswami) for use in electron microscopicvisualization.

C. RecA and mutant RecA803 protein purification

RecA and RecA803 proteins, for use in forming the RecA/probe complexused in the invention, are preferably isolated from overproducingstrains, such as E. coli strains JC12772 and JC15369 (obtained from A.J. Clark and M. Madiraju). These strains contain the RecA codingsequences on a "runaway" replicating plasmid vector present at high copynumbers per cell. The RecA803 protein is a high-activity mutant ofwildtype RecA (Madiraju).

The RecA proteins can be purchased from Pharmacia or purified using fastprotein liquid chromatography (FPLC) on a hydroxylapatite columnfollowed by an anion (Mono®Q) exchange column. The isolation procedurecombines and modifies published procedures (Shibata et al., Griffith).Details are provided in Example 1.

The standard assays for monitoring the protein purification includeassay of 30,000-dalton RecA protein by SDS-polyacrylamide gelelectrophoresis (PAGE) (Pharmacia Phastgel system), enzyme assay ofssDNA-dependent ATPase activity using γ-³² P! ATP and cellulosethin-layer chromatography developed in a solvent of 0.5M LiCl and 0.25Mformic acid, assay of DNase, assay of D-loop activity with 500-meroligonucleotide probe.

Analysis of total protein from JC12772 and JC15369 cell extracts bySDS-PAGE (denaturing conditions) shows that the 30,000-dalton RecAprotein is the major protein produced in these strains.

The SDS-PAGE profiles of the final Mono-Q-purified RecA and RecA803proteins showed a single 30,000-dalton band, free of other cellularpolypeptides as detected by silver staining.

D. Preparation of RecA DNA Probe Complexes

The duplex nucleic acid in the biological structure of interest isreacted with a probe complex composed of RecA protein stably bound tothe single-stranded probe. The complex is preferably prepared in astabilized form in the presence of ATPγS.

RecA protein coating of probes is normally carried out as detailed inExample 2. Briefly, the probe, whether double-stranded orsingle-stranded, is denatured by heating at 95°-100° C. for fiveminutes, then placed in an ice bath for 20 seconds to one minutefollowed by centrifugation at 0° C. for approximately 20 sec, beforeuse. Denatured probes can placed in a freezer at -20° C.; preferably,however, they are immediately added to standard RecA coating reactionbuffer containing ATPγS, at room temperature, and to this is added theRecA protein.

ReCA coating of probe is initiated by incubating probe-RecA mixtures at37° C. for 10-15 min. RecA protein concentration tested during reactionwith probe varies depending upon probe size and the amount of addedprobe, and preferably ranges between about 2 to 25 μM. Whensingle-stranded probes are RecA coated independently of their homologousprobe strands, the mM and μM concentrations of ATPγS and RecA,respectively, can be reduced to one-half those used with double-strandedprobes (i.e. ReCA and ATPγS concentration ratios are usually keptconstant at a specific concentration of individual probe strand,depending on whether a single- or double-stranded probe is used).

E. Probe Hybridization to Permeabilized Biological Structures

According to an important feature of the invention, sequence-specificbinding of the RecA/probe complex to the target duplex contained in abiological structure is achieved by adding the probe complex to thestructure, under non-denaturing conditions, i.e., below the denaturationtemperature of the duplex DNA, and allowing the complex to contact thetarget duplex, typically for 1-4 hours at 37° C., until homologousbinding of the probe complex to the target ONA sequence has occurred.

After probe binding to the target DNA sequence, the target structure iswashed to remove unbound probe complex. In the usual case, where theprobe reporter is a ligand, such as biotin, the washed structure iscontacted with a detectable reporter molecule, such asfluorescence-labeled avidin (FITC-avidin), to bind a detectable reporterto the target-bound probe. The sample material is then further washed toremove unbound reporter molecule. A variety of wash procedures aresuitable. The structure is visualized or otherwise viewed or detected bymicroscopy, fluorescence activated cell sorting, autoradiography, or thelike, as for example described below.

The hybridization condition described in Example 3, for use influorescence-reporter detection of a biotinylated probe, are exemplary.Briefly, between 10-20 μl probe complex is applied to a fixedpreparation on a glass slide. Glass coverslips are placed over thehybridization areas and sealed, and the reactions are incubated in amoist container in a 37° C. 7% CO₂ incubator for between 1-4 hours.Following incubation, the coverslip rubber cement seal is removed andthe slides, with coverslips are washed several times to loosen andremove coverslips and remove unbound probe complex.

The slides are placed in preblock solution, followed by (a) immersion inor application of FITC (fluorescein isothiocyanate)-avidin, in preblocksolution in the dark, then in several washes to remove unboundFITC-avidin. An antifade agent, with or without counterstain such aspropidium iodide, may be used to reduce photobleaching.

If necessary the probe signal may be amplified by reacting the materialon the slide with biotinylated anti-avidin antibody, followed by severalwash steps and addition of FITC-avidin, to enhance the amount offluorescent signal bound to the target duplex.

The target structure is then examined for the presence of thereporter-labeled probe bound to the target nucleic acid, e.g., byfluorescence microscopy or laser scanning microscope.

FIG. 1A shows FITC signal from in situ hybridization of a chromosome Xalpha satellite DNA probe to prepared, isolated HEp-2 cell interphasenuclei fixed on glass slides, in accordance with the present inventionand without amplification, following the protocol detailed in Example 3.Chromosome X is estimated to contain about 5,000 copies/cell of thealpha satellite sequences (ONCOR literature). The biotinylated probe wasreacted and post-labeled with FITC-avidin, as described above.

For comparative purposes, denatured biotinylated chromosome X alphasatellite probe from the same stock used in the FIG. 1A method wascombined with formamide and dextran sulfate under traditional protocolsand was hybridized to HEp-2 cell nuclei using prior art thermaldenaturation (and renaturation) steps, with the results shown in FIG.1B. The procedure required several more hours for total preparation andhybridization time than the FIG. 1A method, involved signalamplification, and generally gave a lower level of fluorescent signalthrough the nuclei.

A second method, reported in Example 4, shows that the method giveshigh-probe target specificity in a low copy number target sequence,without probe signal amplification. In this method, a chromosome-7 alphasatellite DNA/RecA complex is hybridized with HEp-2 interphase nuclei,as above. Chromosome 7 contains about 10 copies of the alpha satellitesequence probe used (ONCOR®probe D7Z2).

FIG. 2A shows the target signal pattern after probe binding and FITClabeling, in accordance with the invention. As seen, the probe islocalized in two distinct spots, presumably corresponding to the twochromosome 7's containing the alpha satellite sequence.

FIG. 2B shows the in situ hybridization probe bound target patternachieved with the same probe, after amplification following prior artmethods described above. Probe localization appears to be less specificthan in the method of the invention. Further, total preparation andprobe hybridization times were many hours longer.

A third method, reported in Example 5, demonstrates the ability tolocalize a target sequence within a nuclear volume relative to othertargeted DNA sequences and/or the nuclear membrane, using a confocallaser scanning microscope (Zeiss LSM-10). In this method, 100% methanolfixed HEp-2 cells were probed in suspension with the RecA/chromosome-Xalpha satellite DNA probe complex, and labeled with FITC-avidin, as inFIG. 1A above. FIG. 3A shows the pattern of probe binding in a dividingnucleus. To localize the bound probe, the same field was viewed by phasecontrast microscopy, without changing the focus of the lens (FIG. 3B).By examining the two photomicrographs, the relative position of thenuclear membrane and nuclear division plane can be seen with respect tothe probe-labeled chromosomes.

The method of the present invention also facilitates the detection ofspecific DNA sequences in metaphase chromosomes using nativeRecA-mediated fluorescence in situ hybridization. RecA coatedbiotinylated probe specific for human chromosome 1 alpha-satellitecentromeric sequences was reacted with fixed HEp-2 cells on slides usingthe native RecA-mediated fluorescence in situ hybridization (Example 6).Before RecA-coated probe mix addition, cells were incubated at 60° C.with 10 mM Tris-acetate (pH 7.5). This incubation step, below thedenaturation temperature of cellular nucleic acid targets, improves theefficiency of the fluorescence in situ hybridization reaction. InExample 6, using this incubation step 73% of all cell nuclei showedfluorescence hybridization signals. FITC hybridization signals werevisualized using a Zeiss LSM in 488 nm argon-ion laser-scanning mode.The FITC hybridization signal is superimposed on the phase image of thechromosomes to identify its position (FIG. 12). Note that the FITC probesignal is, as expected, located at the centromere.

RecA-mediated fluorescence in situ hybridization also facilitates thedetection of unique gone sequences. RecA-coated biotinylated probesspecific for the p53 gene (Oncor) were reacted with fixed cells insuspension using native fluorescence in situ hybridization reactions(Example 7). FITC probe signals were observed with a Zeiss LSM in 488 nmargon-ion laser-scanning mode. Signals were apparent without anyamplification of signal (i.e., extra signal amplification steps). Theresults of this analysis are presented in FIG. 13: FIG. 13A, 13C and13E, FITC hybridization signals; FIG. 13B, 13D, and 13F, phase images ofcells in 13A, 13C and 13E, respectively; FIG. 13A to 13D, HEp-2 cells;and FIG. 13E and 13F, HCC "Alexander" cells. The FITC hybridizationsignals in FIG. 13E are superimposed on the phase image of the cell inFIG. 13F. Note that all hybridization signals are within cell nuclei andthat FITC signals are often seen as pairs indicative of newly replicatedDNA. The cell nucleus in FIG. 13D appears to be in the process ofdividing. The results demonstrate the sensitivity of the method of thepresent invention for detecting unique sequences in solutionhybridization reactions.

In addition to detection of unique sequences in solution hybridizationreactions, the method of the present invention is also effective for thedetection of unique gene sequences using fixed cells on slides.RecA-coated biotinylated p53 probe (Oncor) was reacted with fixed HCCcells on slides using a native fluorescence in situ hybridizationreaction (Example 8). This reaction contained topoisomerase II and wasnot incubated in buffer before probe addition (Example 8). FITC probesignals were observed with a Zeiss LSM in 488-nm laser-scanning mode.Hybridization signals were apparent without any amplification of signal(i.e., extra signal amplification steps). Sample results are presentedin FIG. 14. In FIG. 14: 14A and 14C, FITC signals; 14B and 14D, phaseimages of cells seen in 14A and 14C, respectively. Note that allhybridization signals are within the nucleus and signals often appear aspairs. The position of the signal pairs in the nucleus shown, forexample, in 14A and 14B suggests in this nucleus the signal mayrepresent a stage after DNA replication. These results demonstrate thesensitivity of the method of the present invention for detecting uniquesequences using fixed cells in hybridization reactions.

In addition to the ability of the method of the present invention to beused for the detection of unique cellular gene sequences, the method canalso be used for the detection of unique viral nucleic acid sequences.RecA-coated HBV DNA probes paM6 and "BIOPROBE" were reacted with 100%methanol fixed cells in suspension using a native fluorescence in situhybridization reaction (Example 9). Both probes used in theseexperiments detected HBV sequences in the human HCC cells with highefficiency ("BIOPROBE®", 81%; pAM6, 95%). FITC hybridization signalswere observed with a Zeiss LSM in laser scanning mode. In FIG. 15, theobserved FITC signals from the HBV probes are shown superimposed on thephase images of the cells: 15A and 15B, "BIOPROBE®", 15C-15E, pAM6probe. Note that all signals appear to lie within the nuclear region.Both DNA probes generated multiple FITC hybridization signals in eachHCC cell nucleus. The "BIOPROBE®" signals appear less intense than thepAM6 probe signals. This is likely due to size of the probes used. ARecA-facilitated pairing reaction between single-stranded probe(s) andlinear duplex target DNAs in solution increases in efficiency withincreasing probe strand size: single-stranded "BIOPROBE®" strandsaverage <250 bs and pAM6 single-strands average 300-500 bases in size.This difference might also be due to the fact that the probes containHBV gnenomes of different serotypes ("BIOPROBE®", adr-4; pAM6, adw).These results indicate that the method of the present invention isuseful for the detection of vital DNA sequences. Probes specific for anyvital DNA target of interest can be generated, RecA-protein coated, andused in the in situ hybridization method of the present invention. Inaddition to fluorescent detection a number of other detection methodsmight be used including, not limited to, the following:chemiluminescence (Tropix Inc., Bedford, Mass.) and radioactivity.

The method of the present invention also has a good specificity oftarget detection. The specificity of the present method was examined asfollows. Thirty ng of RecA coated single-stranded biotinylated HBV probewas reacted with ATCC HCC "Alexander" cells using a standard native insuspension fluorescence in situ hybridization protocol (Example 10).

The specificity of the reaction signal for HBV targets was tested byadding 240 ng of either excess RecA-coated single-strandednon-biotinylated homologous DNA, or 240 ng of nonhomologous competitorDNAs (Example 10). Biotinylated HBV probe and non-biotinylated HBV andφ×174 competitor DNAs were nick-translated under the same conditions toinsure that they were of a similar size (average 400-500 bs). Unlabeledhuman placenta DNA, (100-120 bp fragments) was obtained from Oncor("BLOCKIT™ DNA"). The results (Table 1; Example 10) show that onlyhomologous HBV DNA, not heterologous DNAs, specifically competes withthe biotinylated HBV DNA probe signal.

Representative cells from the competition experiments described in Table1 are shown in FIG. 16. In the FIG.: 16A, Biotinylated HBV probe+excessunlabeled HBV DNA; 16B, Biotinylated HBV probe+excess unlabeled φ×174DNA; and 16C, Biotinylated HBV probe+excess unlabeled human placentaDNA. FITC probe signals were observed with a Zeiss LSM in laser scanningmode. The observed FITC signals from the HBV probes are shownsuperimposed on the phase images of the cells. Note that it is clearfrom the signal and cell images that homologous HBV DNA specificallycompetes with the biotinylated HBV DNA probe signal but heterologous DNAdoes not compete. Thus, the RecA-facilitated native fluorescence in situhybridization reaction detects specific nucleic acid targets that arehomologous to labeled probe DNA.

From the foregoing, it will be appreciated how various objects andfeatures of the invention are met. The invention provides a simplifiedand less time consuming procedure(s) for localizing target sequence in abiological structure. The method reduces artifacts by eliminating theneed for a heat denaturation step and by reducing the need for signalenhancement, and allows more rapid and well defined detection of targetsequences, including target sequences of low copy number.

In particular, the method allows detection of low-copy sequences withoutthe requirement to first amplify the sequences. A comparison of FIGS. 2Aand 2B demonstrate that this feature greatly enhances the specificityand resolution of the method over prior art approaches. Since most genemapping and chromosomal studies are expected to involve specificlow-copy sequences, the present method provides an important advantagefor diagnostic gene mapping studies, as well as for diagnosticapplication involving unique or low-copy numbers of various pathogensequences. These later applications are described in Section II below.

Further, the methods described herein are efficacious for the detectionof (i) unique, i.e., single copy, gene sequences, and (ii) unique ormultiple viral nucleic acid sequences, in hybridization reactionscarried out in solution and on slides.

As disclosed in the co-owned patent application for "DiagnosticApplications of Double D-Loop Formation" filed on even date herewith,stable RecA-coated probes prepared from duplex DNA fragments can formdouble-probe hybrid structures with target duplex DNA. Although suchdouble-probe structures have not been shown for probe binding under insitu hybridization conditions, the presence of such structures, ifformed, could be exploited to effectively double the amount of signalproduced at the in situ target site. Further, the two probes could belabeled with different reporter groups, for example, fluorescent probeswith different absorption or emission peaks, so that target sitescontaining both probes could be distinguished from sites containing oneprobe only.

II. Applications

One general application of the invention is for diagnostic use inlocating and visualizing a selected gene or regulatory sequence in achromosome, and/or in a particular region of the chromosome. The targetgene or sequence may be one which (a) generates a selected gene product,(b) is suspected of performing a critical cell-control function, such asthat of a ribosome, an oncogene, or a tumor suppressor gene, (c) isrelated to a repeat sequence, (d) is suspected of containing a geneticdefect which prevents expression of an active gene product, (e) may berelated in chromosome position to a marker probe region with a known mapposition, and/or (f) may represent an integrated or non-integrated viralsequence, such as a DNA-hepatitis virus (e.g., Hepatitis B Virus (HBV)(Ono, et al.; Fujiyama, et al.; Galibert, et al.) in fixed chromatin orfixed virions.

The diagnostic probe used in the method may be obtained, in some cases,from available plasmids, cosmids, viruses or other vectors, such as fromhuman genomic libraries or may be chemically synthesized. Where the geneproduct is available, the probe may be generated by sequencing enough ofthe protein product to generate probes for PCR amplification, andamplifying and tagging the corresponding gene sequence in genomic DNAusing the probes in a PCR format. The amplified gene material can bepurified by electrophoresis and used directly as the probe, or clonedinto suitable vectors, using standard protocols.

In a typical method, the nuclei are derived from cells staged inmetaphase, using well known methods, then fixed and "dropped" on a glassslide to produce a metaphase chromosomal spread. Alternatively, thechromosome material under investigation may be a spread of an isolatedindividual chromosome(s).

FIG. 4A shows a single metaphase chromosome 10 which may be in isolatedform or part of a field containing an entire set of somatic-cellchromosomes. The chromosome contains a known marker region 12 (gene siteM) whose map location on the chromosome is known, and is suspected ofcontaining a gene region of interest. The chromosome preparation on aslide is reacted with the probe complex, indicated at 14 in FIG. 4A, andcomposed of a probe 16 coated with RecA protein, shown by circles at 18,and having biotin groups, indicated by vertical dashes at 26. Reactionof the probe complex with the chromosome material, in accordance withthe invention, leads to homologous binding of the probe to a gene site S(FIG. 4B) which is the target region of interest.

The binding site S may be visualized, for site localization by a varietyof methods. In one method, illustrated in FIG. 4C, a second probecomplex 22 composed of a probe 24 homologous to known region 12 (genesite M) and also containing biotin groups 26 is added to the chromosomepreparation, and allowed to bind to its region of homology. Afterwashing to remove unbound probe, the preparation is reacted with anFITC-avidin reporter 28, to label both sites on the chromosome with afluorescent tag.

When viewed by fluorescence microscopy, a field such as shown at FIG. 4Cis seen, with the two fluorescence points, shown at 30 in FIG. 4C,providing an indication of the distance between the marker and testsequences on the chromosomes.

In another visualization method, shown in FIG. 4D, the chromosomes arelabeled with one or more specific fluorescent dyes, indicated at 32,which give characteristic staining patterns in metaphase chromosomes(Korenberg, Lawrence, 1990). The chromosomes are also labeled with anavidin reporter 34 containing a fluorescent label having a differentfluorescence excitation wavelength from that of the band stainingfluorescent molecule(s). Using fluorescence microscopy, the chromosomesare visualized at one wavelength, as indicated at 36 in FIG. 4D, and thelocation of the probe on the chromosomes site is visualized at a secondexcitation wavelength. Although reaction with one homologue is shown(4D) all homologous sequences would react with probe.

The invention also provides an improved-method for detecting a varietyof chromosomal abnormalities.

FIGS. 5-10 illustrate how the method can be applied to detecting varioustypes of chromosome aberrations. FIG. 5, frame A shows a normalchromosome 40a containing two linked marker regions 42 and 44 on one ofthe chromosome arms. The two regions in the chromosome are hybridizedwith individual probe complexes, in accordance with the invention, thenlabeled with different fluorescent tags. For example, one of the regionsmay be labeled with an avidin-linked fluorescence reporter specificagainst biotin groups on one probe complex, and the second region,labeled with a second fluorescence reporter carried on ananti-digoxigenin antibody specific against digoxigenin groups on thesecond probe complex. The first and second fluorescence reporters areindicated by open and solid circles, respectively in FIG. 5 and inrelated FIGS. 6-10.

When the two regions are examined by fluorescence microscopy, at theappropriate excitation wavelengths, the two regions are localized by twodistinguishable fluorescence spots (indicated by open and solid circles,in frame B). The two spots indicate the relative orientation of anddistance between the two genomic regions in the normal chromosome.

FIG. 6 illustrates, in frame A, a chromosome 40b which differs fromchromosome 40a by a deletion of chromosome region 44. The mutation isseen, in frame B, as a single fluorescence spot at an excitationwavelength corresponding to region 42 only.

FIG. 7 illustrates, in frame A, a chromosome 40c which differs fromchromosome 40a by an insertion between regions 42, 44 in the chromosome.The insertion is evidenced, in the fluorescence microscopy field seen inframe B, by a greater distance between the two fluorescence spots withrespect to the FIG. 5 distance.

FIG. 8 illustrates, in frame A, a chromosome 40d which differs fromchromosome 40a by a duplication of the region 44. The duplication isseen, in Frame B, as a doublet at the excitation wavelength of theregion 44 probe, as indicated.

FIG. 9 illustrates, in Frame A, a chromosome 40e which differs fromchromosome 40a in that a segment containing region 44 has translocatedto a second chromosome 48e. The translocation is evidenced, in Frame B,by widely spaced fluorescence spots. The identity of chromosome 48e maybe determined, as above, by staining the chromosomes with dyes whichform characteristic metaphase banding patterns (or using chromosome 48marker hybridization), as above.

FIG. 10 shows, in frame A, a chromosome 40f which differs fromchromosome 40a in that the segment carrying regions 42, 44 has beeninverted. The inversion is evidenced, in Frame B, by reversal ofpositions of the two fluorescence spots.

FIG. 12 shows the ability of the method of the present invention todetect specific chromosomal DNA sequences in metaphase chromosomes usingnative RecA-mediated fluorescence in situ hybridization. These datasupport the use of the method of the present invention for nativefluorescence in situ hybridization on slides. Example 6 describes thesteps used to generate the metaphase chromosome fluorescence in situhybridization signals represented in FIG. 12 including the following:the preparation of chromosome 1 alpha-satellite probe and HEp-2 cellspretreated with acetate buffer at 60° C. As expected, in FIG. 12, theFITC hybridization signal is located at the centromere. These datasupport that the native RecA-mediated fluorescence in situ hybridizationtechnique can be used to visualize sequence and gene position onnondenatured DNA in fixed chromosomes or chromatin.

FIGS. 13 and 14 show the ability of RecA-mediated native fluorescence insitu hybridization detection of tumor suppressor gene sequences. Thenative RecA-mediated fluorescence in situ hybridization technique can beused to detect and visualize a unique single copy gene sequence in fixedcells in suspension (FIGS. 13A to 13F) and on slides (FIGS. 14A to 14D)without any signal amplification steps. The results show the detectionof unique p53 sequences on chromosome 17 in ATCC HEp-2 and HCC"Alexander" cells (Examples 7 and 8).

FIG. 15 illustrates the ability of RecA-mediated native fluorescence insitu hybridization to detect nucleic acid sequences in ATCC HCC"Alexander" cells in suspension. FIG. 15 (Example 9) shows hybridizationsignals obtained using two different biotinylated HBV probes,"BIOPROBE®" (FIG. 15A to 15B) and pAM6 (FIG. 15C to 15E). Viral targetswere detected in ATCC HCC "Alexander" cells, known to contain HBVnucleic acid sequences, probed using The native fluorescence in situhybridization technique in cell suspension. HEp-2 cells, not infectedwith HBV nucleic acid sequences and probed with the same probes andtechniques, did not show any hybridization signals. These resultssupport the use of the method of the present invention to detectdiagnostically important viral target sequences in HBV-infected humanliver cells.

FIG. 16 demonstrates the specificity of HBV target detection usingnative fluorescence in situ hybridization. The native fluorescence insitu hybridization assay specifically identifies nucleic acid targetshomologous to probe DNA (FIG. 16 and Table 1). This was demonstrated byshowing that biotinylated pAM6 HBV DNA probe hybridization signal isspecifically competed when reactions contain excess homologous unlabeledpAM6 DNA (FIG. 16A) but not when they contain either excessnonhomologous unlabeled φ×174 DNA (FIG. 16B) or excess unlabeled humanplacenta DNA (FIG. 16C). The results of these competition experimentsdemonstrate that native RecA-mediated fluorescence in situ hybridizationsignals, e.g., with HBV probe DNA and HCC cells in suspension, are HBVspecific.

Generally, the RecA-mediated fluorescence in situ hybridizationreactions of the present invention use RecA protein, cofactor, and 1-2hour incubation times. Single-stranded probes in a broad size rangework, including, but not limited to, average sizes of 100-200, 200-400,300-500, 400-600, and up. Typically, size ranges above 100-200 arepreferred and 300-500 are most preferred. Probes coated with RecAprotein can be stored in the freezer for future use: probes stored forup to 7 days have been tested and gave good hybridization signals.

The above described competition experiments have demonstrated that theRecA-mediated native fluorescence in situ hybridization is specific fordetecting homologous nucleic acid sequences. The hybridization reactionis capable of detecting single copy genes and sequences (e.g., p53),multiple copy sequences (e.g., alpha-satellite chromosome 1), anddiagnostically important viral target sequences (e.g., HBV). NativeRecA-protein mediated fluorescence in situ hybridization reactions arein general, more rapid than standard denatured fluorescence in situhybridization assays. Experiments performed in support of the presentinvention indicate that washing in 1.75×SSC after hybridization improvessignal and decreases background.

Some features of the present invention for native RecA-mediatedfluorescence in situ hybridization include the following: nativeRecA-mediated fluorescence in situ hybridization can be used on 1× PBSwashed, 100% methanol fixed (or 70% ethanol fixed) cells in suspension;signals can be achieved with two hours, or less, of incubation withprobe; the reaction is efficient--for example, with 50 ng probe andstandard conditions, the reaction averages between 65-90% of cells withsignal, depending upon the concentration of probe used; the reactionworks with less than 50 ng probe--concentrations of probe in excess of10 ng are preferred; a number of cofactors, including ATPγS, GTPγS, ATP,dATP and a combination of ATPγS and ADP, work in these reactions--oneembodiment employs ATPγS concentrations in the range of approximately0.24 to approximately 2.4 mM (preferred embodiments include the range ofapproximately 0.24 to 0.48 mM); a wide range of RecA monomer:nucleotideratios work well, including 1:1, 1:0.8, 1:2 and 1:2.5 (a preferredembodiment utilizes 1:2); the amount of signal obtained with aChromosome #1 alpha-satellite probe and native RecA-mediatedfluorescence in situ hybridization on slides with HEp-2 cells arecomparable to those obtained using a standard denatured fluorescence insitu hybridization technique; the reaction works in the presence ofaccessory proteins (e.g., single-strand binding protein (SSB),topoisomerase I and topoisomerase II); and when the reactions arecarried out for samples fixed on slides the reaction efficiency isimproved from an average range of 5-20% to 55-80%, by incubating slidesin 10 mM Tris-acetate buffer pH7.5 at 55°-60° C. for 30-45 min beforeadding RecA-coated probe mix. This temperature is below the denaturationtemperature of intracellular nucleic acids.

It will be appreciated that the above applications of the method, to theextent they involve probe binding to a single or small-copy-numbertarget sequence are uniquely suited to study by the present method.

Another general application of the method of the invention is fordiagnostics, typically for detecting changes in chromosome ploidy orrearrangement, or presence of a viral or bacterial or parasitic pathogenin an infected organism, organ, tissue, or cell. This application isspecifically discussed above and is generally illustrated in FIGS.11A-11C for detection of virus infected cells, such as cell 50. Virionparticles (or integrated viral genomes) contained in the cell are shownat 54. The cells. e.g., blood cells, are obtained from the test subject,and treated to permeabilize the cellular structures, as discussed above.To the permeabilized cells (FIG. 11A) is added a virus-specific DNAprobe complex 56, with sequence specific binding of the DNA complex tovirus duplex nucleic acid being followed by addition of a fluorescentmarker molecule 58, for virus-complex labeling (FIG. 11B). The probesignal may be enhanced, if necessary, by the amplification of reporterreagents described above, e.g., a biotinylated anti-avidin antibody,followed by a second fluorescence-labeled avidin reporter molecule.

The labeled cells may be examined by fluorescence microscopy, to detectand localize infecting virus nucleic acid in the cells. Alternatively,cell infection, and percent cells infected, can be determined byfluorescence activated cell sorting (FACS), as illustrated in FIG. 11C.This figure shows a group of blood cells, such as cells 60, 62 passingthrough a capillary tube 64 in a FACS device equipped with a detector 66for detecting fluorescence in individual cells passing through thedetector region. Fluorescence labeled cells are indicated by darkshading in the figure. It is seen that the method provides rapiddetection of infected cells, for diagnostic purposes, and is capable ofmeasuring level of infection and percentage of cells infected. Thus, forexample, the method can be used to assess the progress of an anti-virustreatment, by measuring decreases in cell infection over the treatmentperiod.

The FACS device may be further equipped with sorting apparatus forcapturing fluorescence-labeled cells, to form a concentrate of infectedcells. The concentrate, in turn, can be used as a source of viralnucleic acid, for purposes of identifying and cloning the viral genome.

The following examples, which are intended to illustrate but not limitthe invention, illustrate particular methods and applications of theinvention.

EXAMPLE 1 Purification of RecA Proteins

RecA and RecA803 proteins were isolated from the overproducing strainsJC12772 and JC15369 (obtained from A. J. Clark and M. Madiraju), or RecAwas purchased from Pharmacia.

RecA and RecA803 proteins were purified by modification of publishedprocedures (Shibata, Griffith) involving fast protein liquidchromatography (FPLC) using a hydroxylapatite column (obtained as powderfrom BioRad) followed by an anion ("MONO®Q", Pharmacia) exchange column.

Protein purification was monitored as follows:

(i) identifying the 38,000-dalton RecA protein by SDS-PAGE ("PHASTGEL™"system, Pharmacia, Piscataway N.J.);

(ii) assay of the RecA ssDNA-dependent ATPase activity using γ-³² P!ATPand single-stranded DNA (Shibata). The products of the reaction wereseparated using PEI cellulose thin-layer chromatography Science, NJ):the PEI plates were developed in a solvent of 0.5M LiCl and 0.25M formicacid. Products were detected by autoradiography.

(iii) assay of DNase activity. DNase activity was monitored byincubating the RecA protein samples with a mixture of φ×174 linearizedand supercoiled circular double-stranded RF and circular single-strandedDNAs in RecA strand-transfer buffer (Cheng) for 1 hr at 37° C. DNAnicking and digestion were monitored after deproteinization byvisualizing the DNAs with ethidium bromide after agarose gelelectrophoresis and comparing the quantities of each DNA type in theRecA incubated samples with those incubated in buffer without RecA. OnlyRecA protein samples showing no detectable DNase activity were used.

(iv) assay of D-loop activity with 500-mer oligonucleotide probe using amethod modified from Cheng.

Silver stained SDS-polyacrylamide gel profiles of the final"MOMONO-Q®"-purified RecA and RecA803 proteins showed a single38,000-dalton from each preparation that was essentially free of othercellular polypeptides.

EXAMPLE 2 Preparation of Probe Complex

Biotinylated chromosome X alpha satellite DNA probe was obtained fromONCOR (Gaithersburg, Md.).

Probe diluted in sterile MilliQ® (Millipore) H₂ O was denatured in a 0.5ml microcentrifuge tube in a 100° C. heat block for 5 min, and the tubeimmediately placed in an ice water bath. Approximately 5 min prior toaddition of denatured probe to the hybridization mixture the tubecontaining the probe was placed in ice in a freezer at -20° C. The probehybridization mixture contains the following components in a broad rangeof concentrations and is combined in the order listed: 1 μl of 10× RecAreaction buffer 10× RecA reaction buffer:100 mM Tris acetate pH 7.5 at37° C., 20 mM magnesium acetate, 500 mM sodium acetate, 10 mM DTT and50% glycerol (Cheng)); 1.5 μl ATPγS from 16.2 mM stock (3.24 and 1.62 mMstocks can also be used), (Pharmacia) (rATP, daTP, GTPγS, or acombination of ATPγS and ADP may be used in some reactions); 0.75 μl 20mM magnesium acetate; 4-60 ng (or more in some reactions) of denaturedprobe in sterile ddH₂ O or TE (20 mM Tris HCl, pH7.5, and 0.1 mM EDTA);RecA (when prepared in our own laboratory and the exact amount of μladded varies depending on concentration of stock, when purchased fromPharmacia, 1.25 μl 0.137 mM stock). The mixture was incubated at 37° C.for 10 min followed by addition of 0.5 μl/reaction of 200 mM magnesiumacetate. Final concentrations of reaction components are: 4.0 mM to 10mM Tris acetate, 2.0 mM to 15 mM magnesium acetate, 20.0 mM to 50 mMsodium acetate, 0.4 mM to 1.0 mM DTT, 2% to 5% glycerol, 0.24 mM to 2.5mM ATPγS, 0.005 mM to 0.02 mM RecA.

EXAMPLE 3 In situ Hybridization with Chromosome X Probe

A. Preparation of HEp-2 Cell Nuclei

HEp-2 cells were originally derived from human male larynx epidermoidcarcinoma tissue. HEp-2 is chromosome ploidy variable (Chen).

The cells were cultured for 24 hours after seeding in DMEM (Whittaker orGIBCO-BRL) supplemented with 10% FBS, sodium pyruvate and Penstrepantibiotics mix at 37° C. under standard conditions. The cells werepelleted by low-speed centrifugation and gradually resuspended in 75 mMKCl in a 37° C. water bath, and allowed to incubate for between 5 and 15min for the desired amount of nuclear swelling to occur, followed byaddition of 3:1 ice cold methanol:acetic acid and centrifugation at 6°C.

One ml of fluid was left in the tube with the pelleted cells, additionalice cold methanol:acetic acid was added, and the cells suspended bygentle mixing of the tube, followed by centrifugation. Repeatedadditions of methanol:acetic acid degrades cytoplasm and isolated nucleiwere obtained by repeated additions of methanol:acetic acid followed bymixing and centrifugation as above. (HEp-2 and other cell types may befixed in alternative ways, some of which do not degrade fixedcytoplasmic structures).

Finally, the preparation of nuclei was resuspended in 3:1methanol:acetic acid at a concentration about 2×10⁶ /ml and is eitherdropped by pipette in 10 μl aliquots onto clean glass slides which werestored at -20° C., or the suspended nuclei or cell preparation arestored at -20° C. for later use.

B. Nondenatured Nucleic Acid Target-Hybridization Reaction

Ten μl of probe mixture/reaction from Example 2 was applied to the fixedpreparation on glass slides. Glass coverslips were placed over thehybridization areas and sealed with rubber cement, and reactions wereincubated enclosed in a moist container in a 37° C. CO₂ incubator forbetween 1-4 hours. Following incubation, the rubber cement was removedand the slides were washed in coplin jars 3 times for 10 min each in2×SSC (20×SSC: 3M NaCl, 0.3M sodium citrate, pH 7.0 is used in all SSCcontaining preparations in these assays) in a water bath at 37° C. Otherwash conditions may also be used.

The slides were placed in preblock solution 4×SSC, 0.1% Triton®X-100, 5%Carnation®nonfat dry milk, 2% normal goat serum (Gibco), 0.02% sodiumazide, pH 7.0! for 25 min at room temperature (RT), followed byimmersion in 5 μg/ml FITC-avidin DCS, cell sorter grade (vector, A-2011)in preblock solution for 25 min at RT. The slides were washed in 4×SSC,4×SSC and 0.1% Triton® X-100, and 4×SSC for 10 min each at RT, followedby brief rinsing in double-distilled H₂ O and dried. Antifade wasapplied 100 mg p-phenylenediamine dihydrochloride (Sigma P1519) in 10 mlPBS adjusted to pH g with 0.5M carbonate-bicarbonate buffer (0.42 gNaHCO₃ adjusted to pH 9 with NaOH in 10 ml ddH₂ O) added to 90 mlglycerol, and 0.22 um filtered!, and antifade mounting medium andcoverslips were placed over the preparations. Antifade containing acounterstain such as propidium iodide or DAPI was sometimes used insteadof antifade alone. FIG. 1A shows a fluorescence micrograph of a cellnucleus from the above preparation (no signal amplification).

If necessary, signal amplification may be performed as follows: Slidesare washed for 5-10 min in 4×SSC and 0.1% Triton X-100 at RT to removecoverslips and antifade, followed by incubation in preblock solution forup to 20 min, then are incubated with biotinylated goat anti-avidinantibody (Vector BA-0300) a: a concentration of 5 μg/ml diluted inpreblock solution for 30 mix at 37° C. Slides are washed for 10 min eachin 4×SSC, 4×SSC and 0.1% Triton®X-100, 4×SSC at RT followed byincubation in preblock solution for 20 min at RT, then immersed inpreblock solution with 5 μg/ml FITC-avidin for 20 min at RT. Slides areagain washed in the 4×SSC series, briefly rinsed in dd H₂ O, and mountedwith antifade or antifade with counterstain.

C. Hybridization by Heat Denaturation of the Nucleic Acid Target

For comparative purposes, in situ hybridization by heat denaturation ofnuclear substrate was performed in parallel. Denatured labeled Xchromosome probe was added to the nuclei, denatured on a slide underONCOR protocols. The same nuclear preparations were used as in thenondenatured method. The signal amplification procedure suggested byONCOR was used to enhance the hybridization signal. Thereafter, theslide was maintained at 37° C. overnight. The procedures and materialsgenerally followed that of the ONCOR®Chromosome In situ Kit, Cat No.S1370.

FIG. 1B shows a fluorescence micrograph of a cell nucleus from the abovesignal amplified preparation.

EXAMPLE 4 In situ Hybridization with Chromosome 7 Probe

Biotinylated DNA probe to chromosome 7 alpha satellite DNA was obtainedfrom ONCOR. The probe was denatured and could be stored frozen for atleast five weeks. 32 ng of denatured freshly thawed DNA probe in 16 μl(1:2, probe:H₂ O, 2 ng/μl DNA) were added to the same amount ofhybridization mixture and in the same order given in Example 2.Following incubation of the probe mixture at 37° C. for 10 min and finaladdition of 0.5 μl 200 mM magnesium acetate, the reaction contained atotal of 21 μl.

Probe was incubated on the nondenatured HEp-2 target cell nuclei(Example 3B) for 2.5 hours at 37° C. in a incubator followed by washing,blocking, and FITC-avidin incubation exactly as described for probe tochromosome X in Example 3B. The time to conduct the experiment,including the ethanol series treatment of the slide was approximately 5hours. FIG. 2A shows a fluorescence micrograph of a cell nucleus fromthe treated preparation.

For comparison, the nuclei were reacted with chromosome 7 probe underheat-denaturation conditions, as in Example 3C. Briefly, 5 ng denaturedprobe chromosome 7 alpha satellite DNA was combined with hybridizationbuffer (Hybrisol®V1, ONCOR, as in FIG. 1B) and denatured using ONCORprotocols. 7 μl of the probe mixture was hybridized with HEp-2 cellnuclei for 16 hours and the reaction treated according to ONCORprotocols, including signal amplification. FIG. 2B shows a fluorescencephotomicrograph of the treated denatured nuclei.

EXAMPLE 5 Detection of Specific Chromosome Sequences in Methanol FixedInterphase Nuclei in Suspension

A probe specific for the X chromosome alpha satellite DNA, Oncor probestock (also used in Example 2) was diluted and denatured at 100° C. for5 min, immediately placed in an ice-water bath (for approximately 15min) and stored in a -20° C. freezer briefly (about 5 min) beforeaddition to the hybridization mixture. The hybridization mixture wascombined in the following order (components, concentrations, andmixtures are described in detail in Example 2): 1 μl 10× RecA reactionbuffer (see Example 2), 1.5 μl ATPγS (16.2 mM stock, Pharmacia), 0.75 μlmagnesium acetate (20 mM stock), 12 μl of denatured probe (ONCOR)containing 60 ng in a 1:2 dilution in H₂ O (20 ng or more than 60 ng canalso be used), RecA (0.137 mM stock, Pharmacia). The mixture wasincubated in a 37° C. water bath for 10 min followed by addition of 0.5μl 200 mM magnesium acetate.

HEp-2 cells were fixed in 100% methanol-(or other appropriate solutions)at -20° C. at a concentration of approximately 2.5×10⁶ /ml. About 0.5 mlof the suspended cells (1.25×10⁶) were centrifuged in a "TOMY"centrifuge set at 6° C. in a 1.5 ml microcentrifuge tube and resuspendedfollowed by centrifugation in 200 μl to 1 ml of 70%, 85% and 100% icecold EtOH. After the final centrifugation and removal of 100% EtOHsupernatant the pellet was resuspended in 200-500 μl 1× RecA reactionbuffer at RT, and placed in a 0.5 ml centrifuge tube and centrifuged.

The completed probe mixture was mixed with the pellet, and the tubeplaced in a 37° C. water bath for 1.5-2.5 hours. Incubation was stoppedby addition of 250 μl 2×SSC (prewarmed to 37° C.) followed bycentrifugation. The pellet was resuspended in 2×SSC (prewarmed to 37°C.) and incubated for 5 min at 37° C. Following centrifugation thepellet was resuspended in 500 μl blocking solution at RT far 20 min,then centrifuged and resuspended in 10 μg/ml FITC-avidin in 100 μlblocking solution at RT in the dark, for 20 min. The tube wascentrifuged and 250 μl 4×SSC mixed with the pellet, again centrifuged,and 250 μl 4×SSC with 0.1% Triton®X-100 mixed with the pellet and againcentrifuged with 250 μl 4×SSC all at room temperature. After a finalcentrifugation the pellet was mixed with approximately 20 μl antifade.Specific signal was noted in approximately 30% of the suspended cells.Note: Experiments using 100% methanol fixed whole cells and/or fixednuclei and other concentrations of different washing components haveshown 50-90% reaction.

The FIG. 3A photomicrograph shows a dividing fixed HEp-2 cell nucleus,as viewed with a Zeiss LSM-10 microscope, illustrating the symmetricallylocated FITC-labeled probe-bound centromeric targets. The phase picturein FIG. 3B below was taken of the same nucleus without changing themicroscope focus.

EXAMPLE 6 Detection of Specific Chromosomal DNA Sequences in MetaphaseChromosomes

Biotinylated probe to chromosome 1 alpha-satellite centromeric sequences(pUC1.77: a 1.77 Kbase pair long human EcoRI fragment in the DNA vectorpUC9; Cooke, et al.; Emmerich, et al.) was prepared using the BRLNick-translation System in the presence of bio-14-dATP (Gibco-BRL,Gaithersburg Md.). The nick translations were performed essentially asdescribed by the manufacturer (BRL) with the following modification:twice the recommended amount of enzyme was added and the reaction wasincubated at 15° C. for 1 hr 45 minutes. These nick translation reactionresulted in probes with an average single-strand size of approximately300-400 bp.

Nick-translated probes were precipitated in 0.3M sodium acetate inethanol, resuspended in 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, and the DNAconcentration was determined with the "DNA DIPSTICK™"(Invitrogen).Methanol:acetic acid fixed HEp-2 cells (mostly nuclei; preparedsimilarly to Example 3) mounted on slides were dehydrated by exposure toa series of 70, 85, and 100% cold ethanol incubations. Dehydrated cellson slides were then preincubated in 10 mM Tris-acetate buffer, pH 7.5,at 60° C. for 45 minutes while the RecA-coated chromosome 1alpha-satellite centromeric sequence probe mix was prepared.

The 60° C. preincubation treatment does not denature target DNAs but itdoes improve the efficiency of native RecA-mediated fluorescence in situhybridization reactions performed on fixed cells on slides (from 5-20%to 60-82% improved hybridization). The warmed slide was cooled to 37° C.on a 37° C. surface before prepared probe mix was added to the fixedcell nuclei preparation. Cells were covered with a coverslip and thereaction was sealed with rubber cement.

The DNA probe was heat denatured at 100° C. in 5.16 μl dd H₂ O for 5minutes, quick-cooled in an ice-water bath, centrifuged at 4° C. a"TOMY®" microcentrifuge for 20 seconds to collect the liquid, and thenimmediately added to a mixture containing the other reaction components.Chromosome 1 probe was coated with RecA protein in a reaction mixturecontaining 1 μl of 10× acetate reaction buffer (Cheng et al, 1988), 1.5μl of 16.2 mM ATPγS (Sigma), 0.75 μl of 20 mM Mg(OAc)₂, 0.59 μl of RecA(11.05 μg/μl), 1 μl of DNA probe (50 ng/μl). The total volume ofreaction mix after probe addition was 10 μl. The probe reaction mix wasincubated at 37° C. for 10 minutes and then 0.5 μl of 0.2M Mg(OAc)₂ wasadded. Probe mix was then added to the buffer-treated cell nuclei onslides at 37° C. The reaction was covered with a coverslip, sealed withrubber cement and incubated in a moist chamber at 37° C. for 2 hr.

After cell incubation with probe, the rubber cement was removed and theslide was washed 3× in 1.75×SSC (pH7.4) at 37° C., each wash was 10minutes. The slide was incubated in filtered preblock solution (100 μl)at room temperature for 20 minutes, then with 5 μg/ml FITC-Avidin(Vector, DCS grade) in filtered preblock at room temperature for 20minutes in the dark.

Slides were washed at room temperature 1× in 4×SSC, 1× in 4×SSC+0.1%"TRITON®X-100", and then finally, 1× in 4×SSC. Slides were dipped intoddH₂ O briefly after the last wash and allowed to air dry. Beforecoverslip addition, antifade was added and the cells were observed witha Zeiss LSM.

FIG. 12 shows the hybridization signal from the fixed HEp-2 metaphasechromosomes with the RecA-coated, biotinylated, nick-translated probe tohuman chromosome 1 alpha-satellite centromeric sequences. Under theseconditions, 73% of the cell interphase nuclei including chromosomespreads showed signals. The chromosome alpha satellite specificallyhybridized with the chromosome 1 centromere.

EXAMPLE 7 RecA-Mediated Native Fluorescence In Situ HybidizationDetection of Unique p53 Chromosome 17 Tumor Suppressor Gene Sequences

A. First Conditions: FIGS. 13A and 13B

1.25×10⁶ 100% methanol fixed ATCC HEp-2 (ATCC; American Type CultureCollection, 12301 Parklawn Dr., Rockville Md. 20852) cells were placedin a microcentrifuge tube and put through an ethanol series of 70%, 85%and 100% (Lawrence, 1988 and 1990; Example 5). The cells were pelletedbetween fixation steps. After the 100% ethanol treatment step the cellswere saved as a pellet until just before addition of the 1× acetatereaction buffer wash. All cell centrifugations between steps were for 30seconds at 2.5K in a "TOMY" microcentrifuge.

While probe is incubating with RecA protein, the pelleted cells arewashed in 1× acetate reaction buffer (Cheng et al, 1988). The cells arepelleted again and as much of the buffer wash as possible was removedbefore the addition of the RecA-coated probe reaction mix.

Probe was coated with RecA protein for 10 minutes at 37° C. in a mixcontaining 1 μl of 10× acetate reaction buffer, 0.75 μl of 0.02MMg(OAc)₂, 1.5 μl of 1.62 mM ATPγS (Sigma), 0.59 μl of 11.02 μg/μl RecA,heat denatured probe 5 μl p53 probe (10 ng/μl; Oncor Inc., GaithersburgMd.) and 1.16 μl ddH₂ O!. Before probe addition to washed cell pellet,0.5 μl of 0.2M Mg(OAc)₂ was added to the probe mix. Cells were mixedwith probe and incubated for 3 hr 50 minutes at 37° C.

After incubation, cells were washed 3× in 1.75×SSC pH7.4 (250 μlwashes), then incubated at room temperature for 20 minutes in filteredpreblock, pelleted, and the preblock removed. This step was followed byincubation at room temperature for 20 minutes with 50 μl of filteredpreblock containing 5.0 μg/ml FITC avidin. Cells were washed in 4×SSC,4×SSC+0.1% "TRITON®X-100", 4×SSC, all pH7.4, (250 μl/wash.

A small amount (e.g., approximately 20 μl) of antifade was added to thefinal cell pellet and a portion of the cells were placed on a slide,covered with a coverslip, and observed using a Zeiss LSM. Under thesegeneral conditions, 65% or more of the cells show bright p53hybridization signals (FIGS. 13A and 13B).

B. Second Conditions: FIGS. 13C and 13D

All cells and cell washes were identical to Example 7A. Probe wasreacted with RecA as described above with the exception that the 0.02MMg(OAc)₂ was omitted and 0.75 μl of ddH₂ O was added instead. Underthese conditions, 40% of the cells had bright hybridization signals(FIGS. 13C and 13D).

C. Third Conditions: FIGS. 13E and 13F

All cell washes were identical to Example 7A. Probe was reacted asdescribed above (Example 7A) with the exception that probe coating mixcontained 1.5 μl 16.2 mM ATPγS, the reaction mix was incubated for 13minutes at 37° C. before addition of 0.5 μl 0.2 mM Mg(OAc)₂, RecA-coatedprobe was added to 1.25×10⁶ 100% methanol fixed ATCC HCC "Alexander"cells and reacted for 3 hr 20 minutes at 37° C. Cell washing after probereaction was as described in Example 7A except that cells were reactedwith 50 μl of filtered preblock containing 10.0 μg/ml FITC-Avidin. Underthese conditions, 82% of the cells had bright hybridization signals(FIGS. 13E and 13F).

EXAMPLE 8 RecA-mediated Native Fluorescence In Situ HybridizationDetection of Unique P53 Gene Sequences in HEp-2 Cell Nuclei on Slides

Methanol:acetic acid fixed ATCC HEp-2 cells on slides were reacted withRecA-coated p53 (Oncor) probe. Cells were washed and prepared for probeaddition as described in Example 6 with the exception that the 45minutes incubation with 10 mM acetate buffer pH 7.4 was omitted.

p53 probe DNA coating was done as described in Example 6 except that 1.5μl of 3.24 mM ATPγS, 0.59 μl of 5.51 μg/μl RecA and 0.5 μl containing 2Utopoisomerase II (United States Biochemicals Corp., Cleveland Ohio) wereadded, and half as much denatured probe was added 2.5 μl (25 ng probe)in 3.66 μl dd H₂ O!.

After probe coating with RecA protein, 0.5 μl 0.2M Mg(OAc)₂ was addedand the probe mix was applied to nuclei on slides. Washing conditionsafter reaction with probe were as described for Example 6. Under theseconditions, 20% of the nuclei had bright hybridization signals (FIGS.14A to 14D). The number of interphase nuclei with hybridization signalsin this experiment is less than observed in FIG. 12 (Example 6)--nobuffer incubation step was included in this protocol.

EXAMPLE 9 RecA-Mediated Native Fluorescence in Situ HybridizationDetection of HBV Nucleic Acid Sequences in ATCC HCC "Alexander" Cells inSuspension

1×10⁶ of 100% methanol fixed HCC cells/reaction are placed in 0.5 mlsterile microfuge tubes, centrifuged for 30 seconds at 2K rpm in a"TOMY®" microcentrifuge at 4° C., and the supernatant removed. 200 μl ofice-cold 70% EtOH is added, the treated cells are centrifuged at 4° C.,the supernatant removed, the dehydration step repeated and the samplecentrifuged as above using, sequentially, 85% and 100% iced-cold EtOH.

The cells are centrifuged and resuspended in 200 μl 1× acetate reactionbuffer (same as standard RecA acetate reaction buffer except, minus theglycerol), centrifuged, and resuspended in same 1× acetate reactionbuffer (minus glycerol). Immediately before the addition of the probereaction mixture, the cells are incubated at 37° C. for 10 minutes,centrifuged at room temperature and the supernatant removed.

Biotin-labeled HBV-specific "BIOPROBE®" was obtained from EnzoDiagnostics, Inc. (New York N.Y.). This nick-translated probe isbiotinylated with bio-11-dUTP, contains the whole HBV genome (adr4serotype) and double-stranded probe fragments average 250 bp in size.

A second probe, pAM6, was obtained from the ATCC. pAM6 contains thewhole HBV genome (adw serotype) in plasmid pBR322. pAM6 was labeled withbio-14-dATP by nick-translation with the BRL Nick-translation System asdescribed in Example 6. Heat denatured single-stranded probe averaged300-500 bases in size.

Both HBV probes were coated with RecA protein a 10 μl reactioncontaining 1 μl 10× acetate reaction buffer (Cheng, et al, 1988), 1.5 μl3.24 mM ATPγS, 0.75 μl 20 mM Mg(OAc)₂, 0.53 μl 5.5 μg/μl RecA, and heatdenatured probe 0.23 μl "BIOPROBE®" (60 ng/μl) was in 5.39 μl ddH₂ O; 5μl pAM6 probe (10 ng/μl) was in 1.22 μl ddH₂ O!. Probe coating reactionswere incubated at 37° C. for 10 minutes, then 0.5 μl of 0.2M Mg(OAc)₂stock solution was added and the probe mixes are added to the preparedcell pellets.

The prepared probe mixes were individually added to separate cellsamples and incubated at 37° C. in waterbath for 2 hours. The reactionwas stopped by the addition of 250 μl 1.75×SSC (pH 7.4) at 37° C. Eachsample was mixed, the cells pelleted and the supernatant removed. 250 μlof 1.75×SSC was added and the samples incubated at 37° C. for 5 minutes.This wash was then repeated. The cells were pelleted and to each sample300 μl of filtered preblock was added. The samples were incubated atroom temperature for 20 minutes. The cells were pelleted and thepreblock removed.

To the samples 90 μl of 5 μg/ml FITC-Avidin in filtered preblock wasadded. The samples were incubated at room temperature in the dark for 20minutes. The samples were then pelleted and the supernatant removed. Toeach sample 250 μl 4×SSC was added, the sample mixed gently, and thecells pelleted. The supernatant was removed and 250 μl 4×SSC+0.1%"TRITON®X-100" added. Pellet cells, remove supernatant, add 250 μl4×SSC. The cells were pelleted, supernatant removed, the pellet airdried, and 20 μl of antifade added. The samples were then examined usinga Zeiss LSM.

FIGS. 15A and 15B show the results of the above hybridizations using"BIOPROBE®": 81% of the cells had hybridization signals. FIGS. 15C to15E show the results of the above hybridizations using the pAM6 probe:95% of the cells had hybridization signals.

EXAMPLE 10 Specificity of HBV Target Detection Using Native FluorescenceIn Situ Hybridization in Human HCC Cells Tested by CompetitionHybridization

A. Preparation of probes for competition assay

Both biotinylated and unlabeled pAM6 (ATCC) and φ×174 RFI (New EnglandBiolabs) DNAs were prepared by nick-translation using the BRLNick-translation System. Nick-translation was carried out essentially asdescribed in Example 6, except that reactions for producing unlabeledDNAs contained dATP in place of bio-14-dATp.

Each competition reaction used 1×10⁶ 100% methanol fixed cells andcontained 30 ng of biotinylated pAM6 HBV probe DNA and 240 ng ofcompetitor DNA. Biotinylated HBV probe DNA and unlabeled competitor DNAswere coated with RecA in separate reactions. After RecA coating, theMg⁺⁺ ion concentration of each reaction was adjusted by adding 0.5 μl of0.2 mM Mg(OAc)₂ per 10 μl of coating reaction. Then 10.5 μl ofRecA-coated bio-pAM6 probe (30 ng of DNA) was mixed with an equal volumeof RecA-coated competitor DNA (240 ng). The final volume of each mixtureof RecA-coated biotinylated HBV probe and competitor DNA was 21 μl.

All biotinylated pAM6 DNA was coated with RecA and prepared for use in asingle reaction, 10.5 μl of which was used for each competitionexperiment. Coating of all the biotinylated pAM6 probe in one reactioninsured that there were no differences between reactions other than theDNA competitors. To allow proper RecA coating, both probe and competitorDNA coating reactions contained the same average RecA to nucleotideratio (1 RecA protein monomer: 2 nucleotides).

All the biotinylated pAM6 probe was coated with RecA in a reactioncontaining 4 μl of 10× acetate reaction buffer (Cheng, et al, 1988), 6μl of 3.24 mM ATPγS, 3 μl of 20 mM Mg(OAc)₂, 3.16 μl of 2.2 μg/μl RecA,and 12 μl of 10 ng/μl bio-pAM6 probe (which was heat denatured in 11.84μl ddH₂ O).

Each competitor RecA DNA probe coating mix contained 1 μl of 10× acetatereaction buffer, 1.5 μl 3.24 mM ATPγS, 0.75 μl 20 mM Mg(OAc)₂, 1.25 μl11.05 μg/μl RecA, and either 4.8 μl of 50 ng/μl competitor DNA heatdenatured in 0.7 μl ddH₂ O (non-biotinylated φ×174 or non-biotinylatedpAM6), or 2.4 μl of 100 ng/μl non-biotinylated placenta DNA ("BLOCKIT";Oncor) heat denatured in 3.1 μl ddH₂ O.

All probes were heat denatured at 100° C. for 5 minutes, cooled inice-water approximately 20 sec, spun in a 4° C. microcentrifuge tocollect all the liquid and immediately added to their respective RecAreaction mixture.

Probes were coated with RecA for 15 minutes at 37° C. and then 0.5 μl of0.2M Mg(OAc)₂ was added/10 μl DNA mixture.

B. Reaction Mixtures

The -20° C. stored methanol-fixed cells were prepared for fluorescencein situ hybridization as previously described in Example 9 bydehydrating through a series of cold EtOH washes, followed by 2 timeswashes in 1× acetate reaction buffer (minus glycerol). Cells wereincubated in the last wash buffer for 10 minutes at 37° C. before bufferwas removed and the 21 μl of RecA-coated biotinylated probe andcompetitor DNA mixtures were added to the cell pellets.

Probes were reacted with cells in a 37° C. water bath for 3 hrs.Reactions were stopped by addition of 250 μl 1.75×SSC (pH 7.4) at 37°C., mixed, centrifuged at room temperature (R T) to pellet cells, andsupernatant removed. Cells were washed twice with 250 μl 1.75×SSC at 37°C. for 5 minutes then spun down and the supernatant removed.

300 μl filtered preblock was added to treated, washed cells andincubated at RT for 20 minutes. After centrifugation and supernatantremoval, 90 μl of 5 μg/ml FITC-Avidin in filtered preblock was added toeach reaction, incubated at room temperature for 20 minutes in the dark.FITC-Avidin was removed after cells were pelleted by centrifugation.Reacted cells were washed consecutively in 4×SSC (pH 7.4) mixed gentlywith the cells, 250 μl 4×SSC+0.1% "TRITON®X-100" and 250 μl 4×SSC. Aftereach wash, cells were pelleted and the wash liquid removed.

After the final wash, the cells were air dried and approximately 20 μlof antifade was added to each cell reaction. Cells were mounted onslides, covered with a coverslip and examined with the Zeiss LSM. Cellscontaining moderate to bright hybridization signal(s) were scored aspositive for hybridization (see Table 1).

                  TABLE 1                                                         ______________________________________                                        Specificity of HBV fluorescence in situ hybridization                         in human HCC cells                                                                     # Cells with strong                                                           FITC FISH*             % Cells with strong                           Competing                                                                              Hybridization                                                                              # Cells   FITC FISH Hybridi-                            DNA.sup.a                                                                              Signal       Counted   zation Signal                                 ______________________________________                                        HBV      0            154       0.sup.b                                       φX174                                                                              39           105       37.1                                          Placenta 30            99       30.3                                          ______________________________________                                         .sup.a Nonbiotinylated.                                                       .sup.b 4.5% of these cells showed very faint FITC hybridization. Whereas      FITC signals with the other competing DNAs were easily visible using the      fluorescence microscope alone, the signals with this sample were only         visible when 488 nm argonion laser illumination was used.                     *fluorescence in situ hybridization.                                     

The results presented in Table 1 show that only homologous HBV DNA, notheterologous DNAs, specifically competes with the biotinylated HBV DNAprobe signal.

The cells shown in FIGS. 16A to 16C are from the competition experimentsdescribed in Table 1. In FIG. 16: 16A, Biotinylated HBV probe+excessunlabeled HBV probe DNA; 16B, Biotinylated HBV probe+excess unlabeledφ×174 DNA; 16C, Biotinylated HBV probe+excess unlabeled human placentaDNA ("BLOCKIT™" Oncor). FITC probe signals were observed with a ZeissLSM in laser scanning mode.

The observed FITC signals from the HBV probes are shown superimposed onthe phase images of the cells. Several cells from each experiment areshown. It is clear from the signal and cell images that homologous HBVDNA specifically competes with the biotinylated HBV DNA probe signal butheterologous DNA does not compete.

Although the invention has been described with respect to particularprotocols and applications, it will be appreciated that a variety ofchanges and modifications may be made without departing from theinvention.

It is claimed:
 1. A method of identifying the presence of a known targetsequence in a double-stranded nucleic acid contained in a fixed cellularor subcellular biological structure, in a defined morphologicalrelationship with the structure, comprisingadding to the structure, aprobe complex composed of RecA protein stably bound to asingle-stranded, reporter-labeled probe which is complementary to aduplex target sequence, under conditions in which the complex cancontact the duplex nucleic acid target, allowing the complex to bind tothe target sequence under non-denaturing conditions, removing unboundcomplex from said structure, and examining the structure for thepresence of the reporter-labeled probe bound to the nucleic acid.
 2. Themethod of claim 1, wherein the complex is stabilized by the presence ofa cofactor selected from the group consisting of ATPγS, GTPγS, ATP, dATPand a combination of ATPγS and ADP.
 3. The method of claim 1, whereinsaid probe is labeled with a ligand reporter, and said examiningincludes adding to the structure, specific ligand molecule, includingantibodies, effective to stably bind to said ligand, and having adetectable reporter group.
 4. The method of claim 1, for detecting thepresence in a host cell, of a pathogenic (foreign) target duplex nucleicacid sequence, wherein said complex is added to the cells underconditions of host cell fixation, and said examining includes detectingthe presence of a probe-bound reporter in said fixed cells.
 5. Themethod of claim 1, wherein said examining includes detecting afluorescent reporter bound to the reporter-labeled probe bound to thenucleic acid using either microscopy or a fluorescence activated cellsorter.
 6. The method of claim 1, for localizing a selected targetduplex nucleic acid sequence integrated into a host-cell genome, whereinsaid complex is added to the chromosomes of the cell, and said examiningincludes examining the chromosomes microscopically to determine therelative position of reporter-labeled probe in relation to chromosomeultrastructure.
 7. The method of claim 6, wherein said chromosomes arelabeled with one fluorescence reporter, said probe is labeled with asecond fluorescence reporter, and said examining includes viewing thecells by fluorescence microscopy separately at wavelengths effective toexcite fluorescence in each of the two reporters.
 8. The method of claim6, for localizing the target sequence in a selected chromosome, whichfurther includes adding to the structure a second probe complex composedof RecA protein stably bound to a single-stranded, reporter-labelednucleic acid probe which is complementary to a duplex strand in a knownregion of the selected chromosome, and said examining includesdetermining the relative positions of reporters associated with each ofthe two complexes.
 9. The method of claim 8, wherein the first-mentionedcomplex and the second complex are labeled with different fluorescencereporters, and said examining includes viewing the cells by fluorescencemicroscopy separately at wavelengths effective to excite fluorescence ineach of the two reporters.
 10. The method of claim 1, which furtherincludes amplifying the target duplex nucleic acid in the structureprior to said adding.
 11. The method of claim 1, which further includesamplifying the probe bound to the target by addition of polymerase, andall four deoxytrinucleotides, where one of the deoxytrinucleotidesincludes a reporter label.
 12. The method of claim 1, where said fixedstructures are in solution or on a slide.
 13. The method of claim 1,where said fixed structures are incubated in 10 mM Tris-acetate buffer,pH 7.5, at 55°-60° C. before the addition of said RecA probe complex.14. The method of claim 1, where said allowing the complex to bind tothe target sequence under non-denaturing conditions is carried out forless than 2 hours.
 15. The method of claim 1, where said adding includesthe addition of accessory proteins selected from the group consisting oftoposiomerase I and topoisomerase II.
 16. A method of identifying thepresence of a known double-stranded viral nucleic acid target sequencecontained in a fixed cellular or subcellular biological structure,comprisingadding to the structure, a prone complex composed of RecAprotein stably bound to a single-stranded, reporter-labeled probe whichis complementary to the double-stranded viral nucleic acid targetsequence, under conditions in which the complex can contact thedouble-stranded nucleic acid target, allowing the complex to bind thetarget sequence under non-denaturing conditions, removing unboundcomplex from said structure, and examining the structure for thepresence of the reporter-labeled probe bound to the nucleic acid. 17.The method of claim 16, where the known viral target is a sequencederived from hepatitis B virus.
 18. The method of claim 16, where thefixed structures are incubated in 10 mM Tris-acetate buffer, pH 7.5, at55°-60° C. before the addition of the RecA probe complex.
 19. The methodof claim 16, wherein the complex is stabilized by the presence of acofactor selected from the group consisting of ATP S, GTP S, ATP, dATPand a combination of ATP S and ADP.
 20. The method of claim 16, whereinsaid probe is labeled with a ligand reporter, and said examiningincludes adding to the structure, specific ligand molecule, effective tostably bind to said ligand, and having a detectable reporter group. 21.The method of claim 20, wherein said ligand reporter is digoxigenin orbiotin and said ligand molecule is selected from the group consisting ofan antibody, avidin and streptavidin.
 22. The method of claim 16,wherein said examining includes detecting a fluorescent reporter boundto the reporter-labeled probe bound to the nucleic acid using eithermicroscopy or a fluorescence activated cell sorter.
 23. The method ofclaim 16, for localizing a selected target duplex nucleic acid sequenceintegrated into a host-cell genome, wherein said complex is added to thechromosomes of the cell, and said examining includes examining thechromosomes microscopically to determine the relative position ofreporter-labeled probe in relation to chromosome ultrastructure.
 24. Akit for the practice of the method of claim 16, comprising aRecA-protein coated single-stranded and reporter labelled DNA probederived from the viral nucleic acid sequences, means of removing unboundcomplex from said structure, and means of examining the structure forthe presence of the reporter labelled probe bound to the nucleic acid.25. The kit of claim 24, where the probe is derived from hepatitis Bvirus sequences.
 26. The kit of claim 24, where the reporter is biotinor digoxigenin.
 27. The kit of claim 24, where the kit further includesmeans of detecting the binding of the probe to the known double-strandedviral nucleic acid sequences in a sample and said means of detectionincludes detecting a fluorescent reporter bound to the reporter-labeledprobe bound to the nucleic acid using either microscopy or afluorescence activated cell sorter.
 28. The method of claim 16, wheresaid fixed structures are in solution or on a slide.
 29. The method ofclaim 16, where said allowing the complex to bind to the target sequenceunder non-denaturing conditions is carried out for less than 2 hours.30. The method of claim 16, where said adding includes the addition ofaccessory proteins selected from the group consisting of toposiomerase Iand topoisomerase II.
 31. A method of detecting a single copy nucleicacid target sequence contained in a cellular or subcellular biologicalstructure, comprisingfixing the cellular or subcellular biologicalstructure, adding to the structure, a probe complex composed of RecAprotein stably bound to a single-stranded, reporter-labeled probe whichis complementary to the single-copy nucleic acid target sequence, underconditions in which the complex can contact the nucleic acid target,allowing the complex to bind to the target sequence under non-denaturingconditions, removing unbound complex from said structure, and examiningthe structure for the presence of the reporter-labeled probe bound tothe nucleic acid.
 32. The method of claim 31, where said fixing is insolution or on a slide.
 33. The method of claim 31, where said fixingincludes incubation of the fixed structures in 10 mM Tris-acetatebuffer, pH7.5, at 55°-60° C.
 34. The method of claim 31, where saidallowing the complex to bind to the target sequence under non-denaturingconditions is carried out for less than 2 hours.
 35. The method of claim31, where said adding includes the addition of accessory proteinsselected from the group consisting of toposiomerase I and topoisomeraseII.
 36. The method of claim 31, wherein the complex is stabilized by thepresence of a cofactor selected from the group consisting of ATPγS,GTPγS, ATP, dATP, and a combination of ATPγS and ADP.
 37. The method ofclaim 31, wherein said probe is labeled with a ligand reporter, and saidexamining includes adding to the structure, specific ligand molecule,effective to stably bind to said ligand, and having a detectablereporter group.
 38. The method of claim 37, wherein said ligand reporteris digoxigenin or biotin and said ligand molecule is selected from thegroup consisting of an antibody, avidin and streptavidin.
 39. The methodof claim 31, wherein said examining includes detecting a fluorescentreporter bound to the reporter-labeled probe bound to the nucleic acidusing either microscopy or a fluorescence activated cell sorter.
 40. Themethod of claim 31, for localizing a selected target duplex nucleic acidsequence integrated into a host-cell genome, wherein said complex isadded to the chromosomes of the cell, and said examining includesexamining the chromosomes microscopically to determine the relativeposition of reporter-labeled probe in relation to chromosomeultrastructure.
 41. A kit for the practice of the method of claim 34,comprising a RecA-protein coated single-stranded and reporter labelledDNA probe derived from the single copy nucleic acid sequences, means ofremoving unbound complex from said structure, and means of examining thestructure for the presence of the reporter labelled probe bound to thenucleic acid.
 42. The kit of claim 41, where the probe is derived fromp53 tumor suppressor gene sequences.
 43. The kit of claim 41, where thereporter is biotin or digoxigenin.
 44. The kit of claim 41, where thekit further includes means of detecting the binding of the probe to thesingle copy nucleic acid sequences in a sample and said means ofdetection includes detecting a fluorescent reporter bound to thereporter-labeled probe bound to the nucleic acid using either microscopyor a fluorescence activated cell sorter.